Inspection
of the Safety Assessment of Genetically Modified, the Roundup Tolerant Soybean:

Monsantofs
Dangerous Logic as seen in the Application Document submitted to Japan.

by:
Masaharu Kawata

(Assistant
Professor, School of Science, Nagoya University, Japan)

Mandatory labeling of genetically modified food became effective this
April in Japan. Japanese consumers
can then be able to choose non-genetically modified food by the label in any
shop and store. But,there would be
consumers who dare not to choose it for their confidence in Health and Welfare
Ministry's safety approval or who purchase processed food containing
genetically modified ingredient without knowing it.Safety assessment of genetically modified crop whose unknown
risk with artificially modified gene is raised cannot be too cautious. This is the report of inspection carried
out on Safety Assessment Application Documents submitted by JAPAN- MONSANT for
their herbicide torelant soybean that was approved as food by the Japanese Ministry
of Health and Welfare and as animal feed by the Ministry of Agriculture in
1996.

(1) Information disclosure is but nominal

Application documents to the Ministry of Health and Welfare is kept by
Food Safety Association, one of the extra-governmental organization, and is made
available for inspection in their Tokyo and Osaka offices. However, inspection is only allowed 10am
to 12am and 13pm to 16pm 3 days a week, and no copying or photographing is
permitted.Transcription by hand is
the only method we could take for accurate scrutiny as was in Edo-Samurai
period in old Japanese times. The
application submitted by Monsanto for "Roundup Ready soybean"
consists of 10 volumes, which pile up to 1 meter high. Moreover, the third section on are all
in English. It was impossible for
us to transcribe all of them in given time. We were all told 40 people in 10 days and managed more than
500 pages focusing on important points for best possible effectiveness.When information disclosure law enacted
this April, we must watch closely whether it would make things easier or even
harder with excuse of company secrets and keep the decision making of safety
assessment to a few bureaucrats and their academics. Unless safety assessment is made open to public, concerns over
genetic engineering will be even worse.

(2) What is herbicide resistant soybean
by Monsanto?

In growing soybean, well-planned weed and pest control is important to
get desired harvest. Low input
cultivation becomes possible if soybean itself has herbicide resistance and dust
cropping is done without complexity. Monsanto has endeavored, in vain, till 1990s to achieve this
goal by creating soybean mutant, which is resistant to their best selling organic-phosphate
herbicide Roundup in which the glyphosate is its active ingredient.The resistant strains created had seriously
damaged enzymatic activity of EPSPS(5-enol-pyruvylshikimate-3-phosphate synthase
: one of the enzymes work to synthesize aromatic amino acid, Tyrosine, Phenylalanine,
Tryptphan) and caused growth defect of soybean itself.The genetic engineering technology was
becoming popular then, and was naturally selected tried to introduce gene from
different organism to soybean.Purported herbicide resistant bacterium was found in the sewage water of
the glyphosate factory of Monsanto in USA. This Agrobacterium tumefaciens named as CP4 strain is a kind
of soil bacterium, which could synthesize aromatic amino acid in the presence
of glyphosate.The amino acids sequence
of the enzyme is largely different from that of any plants, and is called class
II EPSPS (refer to as CP4EPSPS hereafter).

The bacterial gene generally can not work in plant cell by just
inserting it to the genome, because the genetic switch called promoter of prokaryotes
and that of eukaryote is different. Then a powerful promoter from "Cauliflower Mosaic
virus" called 35S promoter was connected to the target gene. Next gene engineering was to connect a
small protein called "signal peptide" which carries the CP4EPSPS protein
to where the enzyme is supposed to function, in this case chloroplast.This gene of signal peptide was taken
from flower petunia. A part of plant
cancer virus gene called NOS, which make a signal to stop gene read through is
also required. Thus created
"Roundup tolerant soybean gene" is a completely artificial gene that
never exists in natural kingdom (figure 1) which would be never existed in
natural evolution.

In addition to
these modifications of the genetic construct, Monsanto artificially had to change
genetic codons for efficient translation of the CP4ESPS gene in soybean plant. The 239 nucleotides out of total
of 1,365 (17.51%) were manually converted to other bases (though mostly in the
third letter) in order for the protein synthetic machinery of soybean cell to
decipher the bacterial gene across the species barrier.Thus, the Roundup Tolerant soybean came
to possess a gene unlike either the prokaryotic gene or the eukaryotic gene. It is with reason that gene
modified plants are called "the Frankenstein plants" in Europe. Focal point of safety assessment is
whether such soybean with artificially modified gene is the same as the
conventional one.

(3) A mystery of "The samples used
for analysis and animal dietary test were cultivated without herbicide
application".

The Roundup
Ready soybean marketed is usually applied with the herbicide Roundup. But surprisingly enough, our inspection
revealed that both the gene modified soybean 40-3-2 strain and conventional
strain A5403 were NOT sprayed with Roundup herbicide in their cultivation.Monsanto produced only small amount
sample with Roundup on the side to test residual glyphosate in the harvested
forage, hay and seed.All the
soybean of a few thousand kilograms used in safety experiments was harvested not
sprayed.The reason is not stated
in the documents.

The data obtained with such samples may be therefore not valid to guarantee
safety of soybean that human and animals take in the real life, not just
because of the residue glyphosate is a toxin to kill plants by inhibiting plant
enzyme EPSPS. Effects on other
metabolic pathway must be taken into account particularly when such artificial
genes are inserted. For consumers,
the test results using different sample than marketed soybean may be
meaningless.

(4) Incomplete analysis of introduced protein
CP4EPSPS.

It is expected that CP4EPSPS protein expressed in the bio-engineered
soybean have the same amino acid sequence as the soil bacterium from which the
gene was originated. This can only
be verified when soybean produced protein is isolated and the amino acid sequence
is determined, because exchanging genes between bacteria and higher organism
can sometimes result in partial amino acid change and/or post-translational
modification after expression.Before
inspection we presumed that amino acid sequence of soybean CP4EPSPS was
determined.However, to my
surprise, it was not.

What Monsanto has determined was only 15 amino acids from N-terminal of the
protein, which was expressed in E.coli. The rest of the sequence was presumed one from the nucleotide
sequence of the bacterial DNA. Then only 3.3% of expected total of 455 amino acids was
decided, and the protein is not of soybean!ELISA test described in the documents is the only method to
verify antigenic equivalence of proteins. But antigenic similarity itself does not prove the amino acid
sequences are the same. The true
face of CP4EPSPS protein in the soybean that we are taking is still unknown.

(5) The E.coli expressed protein is used
for acute toxicity test too on rat.

CP4EPSPS protein used for acute toxicity test on rat is also
came from that produced by E.coli harboring CP4EPSPS plasmid. Monsanto excuses in the application
document that to obtain large amount of CP4EPSPS protein from soybean is
difficult. This is unacceptable
because there is a possibility that the inserted gene work differently in
soybean than was in the original bacterium, and therefore the expression product
may be different from that of soybean. Moreover, according to the application document, 0.238mg of
CP4ESPS protein is detected in one gram of genetically modified 40-3-2 soybean,
which is enough concentration to extract without problem. This again is the typical "All for
the conclusion" approach by Monsanto. This kind of problem could be resolved if all CP4ESPS amino
acid sequence in soybean had been analyzed and confirmed equal as the
bacterium. The experiment looks
like conducted on the presumption that the other soybean proteins are the same
as the non-GM soybean as long as the CP4EPSPS is not toxic. If so, this is too easy and one-sided
approach. The core of this problem
is whether the soybean gene gets affected from insertion of foreign gene or
not.The series of experiments described
is incoherent on the fundamentals.

Animal feeding test
is important for safety assessment.Then Monsanto conducted the experiments for animals as rat, cow, chicken,
catfish and quail. However, the
scale of experiment is less than adequate.

For example, in rat experiments, raw and
toasted soybean both genetically modified and non-modified were fed to mere 10
rats each group and feeding period is only limited to 28 days. Toxicity across generation or chronic toxicity
may not be detectable by these limited experiment size and duration.

Under these insufficient experiments however, the data for body and
organ weight of lever, kidney and testicles show obvious difference in the male
rats between both groups, wild A5403 and bio-engineered 40-3-2 soybean.

Raw soybean fed group showed no difference. But toasted soybean 40-3-2
fed male group weighed 6.7% less body weight than A5403 fed group and 13% less
than commercial feed mix fed group at the end of test periods, 28 days. Though this difference is described as
statistically significant in the data sheet, the conclusion ignores these
results and states that "no statistical significance is observed."

The experiments are far from satisfactory in its sample size and the
statistic method used. Our group transcribed all raw data and redid statistical
analysis using Turkey multiple method. The result again showed the apparent
growth obstacle for the body and kidney weight in male rats group fed with
toasted 40-3-2 soybean. I wondered
why there is no such difference in female rats group. The answer to this question seemed to be
the amount of the feed intake where male took 25-30g/day, female rats took only
18-20g (approx. 70% of male)/day. It
is highly possible that female rats also showed significant growth difference
if experiment is conducted in much larger scale and with longer feeding period.

(7) Misguided interpretation and
disregard of data in chemical analysis.

Chemical analysis of the components from both normal and genetically
modified is important to certify so-called substantial equivalence.

We found a highly intended misinterpretation ignoring obvious data difference
between A5403 and 40-3-2 hybrid in the documents. Analysis of raw soybeans showed no difference between gene modified
30-4-2 and non-modified A5403 soybean. Difference is observed in toasted soybeans. Besides such main components like water
content, protein, fat, fiber and ash, the analysis detected trypsin-inhibitor, lectin
and urease which are called harmful physiologically active substance as
feed.Urease is used an indicator
of protein denaturation by heat treatment.

Obvious difference appeared when after toasted with actual feed
processing condition (108,
30min). The concentration of total
protein and potassium were not changed, but concentration of trypsin-inhibitor,
urease, and lectin have significantly higher in the toasted 30-4-2, the
glyphosate-tolerant bean compared to that of A5403 normal bean. These physiologically active substances
remained active even after heat treatment in the genetically modified soybean,
though those of herbicide sensitive normal bean were easily denatured and
inactivated. The high activity of
these elements does not usually satisfy the feed standard.

Monsanto took this result as "only the modified soybeans are
toasted insufficiently in the experiment", and returned and asked re-treatment
of the sample to Texas A & M who processed the beans.Monsanto ordered the condition of
re-toast as 220 
for 25min, which is considerably higher than normal processing of 100,10
minutes. However re-toasting
further widened the difference in the activity between the two strains. Another hybrid 61-67-1,which is another
genetically modified soybean inserted with bacterial CP4EPSPS, showed highly
resistant property to the heat.

Scientist would usually conclude in such case that there is substantial
difference between the two.But Monsanto
dared to challenge this common sense, and concluded again the second toasting
is still not enough. In the end,
they toasted twice further and got the result they wanted, all proteins were denatured
and inactivated. With this result, they concluded that genetically modified and
non-modified soybeans have equivalent properties.

No protein can withstand repeated heat treatment and stay active. This is a common knowledge of protein
chemistry. The argument at normal
feed processing condition is required and no more, no less. Monsanto based their argument on
their presumption " they can't be different" and their need "there
shouldn't be difference". Their
translation of the experiment is "Safe is the conclusion" attitude
and not at all scientific. The
English data volume did not show analysis data of third and fourth heat
treatment, but the Japanese summary volume, as if there were data, has a graph
showing after loss of activity and described that "data from insufficient
heat treatment is not adopted" and "No substantial difference
observed." If you review only
Japanese summary volume and not look into English data volume, you would be
ushered to the conclusion of "Safe."

However we could found in the first and the second analyses data of
toasted soybean a fact indicative of regular heat treatment. Granulated
soybean, when heated, lose weight as water and other volatile components
evaporate, and as the result, relative concentration of non-volatile substance
such as total protein and ash increases. The data shows clearly that the gene modified 40-3-6 and
61-67-1 and non-modified A5403 gone through same level of heat treatment. The decrease of water content also certify
this facts.

(8) Residual herbicide in crop increase,
then the safety standard should be changed high level, Monsantofs conclusion.

By adopting the Roundup tolerant soybean, the herbicide concentration
will rise in the soybean plant and seed, because the herbicide is directly
sprayed on the plant by postemergence application before harvest.The Monsanto studied in detail
how will be the results by changing factors like spraying times, concentration
of the active ingredient glyphosate, duration of harvest after spraying, and
cultivation places.The data show
clearly that the concentration of glyphosate and AMPA (a degraded substance of
glyphosate) in forage and hay increase greatly by postemergence application of
the herbicide compared to that of conventional preemergence application,
although the residual concentration in the plant differed from place to
place.The largest value of the
combined glyphosate and AMPA was 40.187 ppm in forage which is higher than the
US safety standard of 15 ppm in forage and hay in 1994 when FDA and USDA
accepted the application documents.The maximum combined concentration of glyphosate and AMPA in soybean
seed was 13.178 ppm, which is less than 20 ppm of the US standard at that
time.The residual concentration
increased in accordance of the application increased from twice to three
times.Then
cultivating Roundup ready soybean may sometimes violates the US safety
standard.We found a surprising
description in the document to dissolve the problem.

In final conclusion, Monsanto say that gthe maximum combined glyphosate
and AMPA residue level of approximately 40 ppm in soybean forage resulting from
these new uses exeeds the currently established tolerance of 15 ppm.Therefore, an increase in the combined
glyphosate and AMPA tolerance for residues in soybean forage will be
requested.hThey know very well
that adoption of herbicide tolerance crop needs higher safety standards.In effect, the US tolerance standard of
combined glyphosate and AMPA in soybean forage was changed to 100 ppm after
they approved the genetically engineered soybean.

As to Japanese government, they revised the safety standard of combined
glyphosate and AMPA in soybean seed to 20ppm in April 2000, from old standard
of 6 ppm according to the request of US government.Of course Japan become could import soybean from USA without
worrying about of violation of the law by this decision.

Thus, Monsanto, in their rush to verify safety, patch worked
the results of experiments and analyses that are full of voids like a puzzle and
asserted safe with manipulation of the results. They requested, if necessary, even the revision of safety
standard.We found the facts
showing inadequate and incomplete safety assessment described above in the
application document by Monsanto even in our limited work under difficult
situation. The process of genetic recombination
and the results of other animal experiment remained not inspected yet.

Monsanto informed US soybean importing countries in May 2000
that they found Roundup resistant soybean has two extra fragments of the
CP4EPSPS gene in the genome.They
were there since the first FDA approval in 1992, and all the GM soybeans
supplied worldwide contain this gene fragments. Monsanto asserts that these fragmented genes do not create
unknown protein since they have any open reading frame or termination signal
around them. But such basic facts
comes to light 8 years after the approval is a sure indication of how
incomplete the genetic recombination of crop is, and how dangerous safety
assessment can be to rely only on companyfs information and data. We doubt it very much if at all government
experts in charge at the Japanese Ministry of Health and Welfare for safety
assessment had a good sense to have concluded as safe on the bases of such
incomplete application.

The safety assessment of the Monsanto Roundup ready soybean
needs to be reassessed, is our conclusion.